The objectives of the proposed research are to construct alphavirus replicon particle vaccines against Venezuelan, eastern and western equine encephalitis viruses (VEE, EEE and WEE), and perform in vitro characterization, production scale-up, GMP manufacture, and benchmark preclinical studies of these vaccines in preparation for future clinical trials. VEE, EEE and WEE viruses are highly infectious by the aerosol route, cause febrile diseases and encephalitis in animals and humans, can be grown to high titer and stabilized by lyophilization, and are highly credible threats for use in biowarfare or bioterrorism. Existing investigational vaccines have significant shortcomings, including inadequate immunogenicity and undesirable side effects. We propose an alternative approach, using a single-cycle RNA replicon vector system, derived from an attenuated strain of VEE, to produce virus-like replicon particles (VRP) expressing envelope glycoproteins of VEE, EEE or WEE. When inoculated into animals, these VRP vaccines are expected to induce a neutralizing antibody response against the expressed envelope glycoproteins that will confer protection against challenge with VEE, EEE or WEE. Successful preliminary studies have demonstrated the safety and feasibility of this approach. The project will include seven specific aims. (1) Construct VRP vaccines expressing envelope glycoproteins of VEE, EEE and WEE. (2) Perform in vitro characterization of these vaccines. (3) Evaluate safety, immunogenicity and efficacy in murine challenge models. (4) Evaluate safety, immunogenicity and efficacy in primate challenge models. (5) Perform scale-up development of processes for GMP-compliant manufacture. (6) Perform GMP manufacture. (7) Perform benchmark studies to enable moving these vaccines into future Phase I clinical trials. To achieve these aims, the glycoprotein genes from VEE, EEE and WEE will be inserted into self-replicating RNAs (replicons) derived from an attenuated strain of VEE. Using a novel DNA helper system that precludes replicon/helper recombination, the replicons will be packaged into VRP with a VEE glycoprotein coat that confers dendritic cell tropism. Safety, immunogenicity and efficacy will be evaluated using established animal models. Process scale-up, GMP manufacture, and benchmark preclinical testing will be performed based on previous experience with a VRP vaccine for HIV. Results of these studies will provide the basis for future clinical trials. [unreadable] [unreadable]